Studies of the Mechanisms Causing Pancreatitis in Severe HypertriglyceridemiaBjorn Christophersen1*, Randi Sorby2 and Knut Nordstoga3

1Professor of Clinical Biochemistry, Institute of Clinical Medicine, University of Oslo, Norway2Assistant Professor of Veterinary Pathology, Norwegian University of Life Sciences, Oslo, Norway3Professor Emeritus of Veterinary Pathology, Norwegian University of Life Sciences, Oslo, Norway*Corresponding author: Bjorn Christophersen, Professor of Clinical Biochemistry, Institute of Clinical Medicine, University of Oslo, Norway, Tel: 4793899474; E-mail:bjorn.christophersen@medisin.uio.no

Rec Date: October 28, 2014; Acc Date: December 15, 2014; Pub Date: December 21, 2014

Copyright: © 2014 Christophersen B, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

Hypertriglyceridemia has been estimated to cause around 7% to 10% of the total number of cases of acutepancreatitis in man. The mechanisms involved in the progression of hypertriglyceridemia to pancreatitis are not wellunderstood.

In this paper, we refer to different mechanisms proposed by previous authors to explain the pathogenesis. Wediscuss these theories in relation to our own experimental results. It has been difficult to verify previous theoriesbecause pancreas biopsies cannot easily be obtained in humans in the early, mild stages of acute pancreatitis. Inpatients with severe pancreatitis, the pancreas is typically grossly pathological. We have used an animal model withlipoproteinlipase deficiency. In these animals, severe hypertriglyceridemia can be induced by feeding a high-fat diet,which in turn induces the development of severe pancreatitis. By sacrificing animals at different times, it waspossible with the use of light- and electron microscopy to monitor the development of acute pancreatitis from theearliest detectable changes to the most advanced, full-blown stages.

We found that the earliest detactable changes consisted of a selective degeneration of the mitochondria in theexocrine cells. At the same early stage the other intracellular structures, including the endoplasmatic reticulum, werewell preserved. Mitochondria are known to be the major source of cellular free radicals liberated by the electrontransport chain which may lead to oxidative stress. In normal cells antioxidant mechanisms such as vitamin E,glutathion peroxidase and others will neutralize free radicals and thus prevent oxidative stress. Our finding of aninitial mitochondrial degeneration, probably caused by free fatty acids deranging the mitochondial function, supportthe view that antioxidants may have a role in the prevention of recurrent hypertriglyceridemia-induced pancreatitis.

Keywords: Acute pancreatitis; Chronic pancreatitis;Hypertriglyceridemia; Hyperlipemia

Abbreviation:FFA: Free Fatty Acids

IntroductionThe detailed pathophysiological mechanisms involved in the

progression of hypertriglyceridemia to pancreatitis are little knownand little studied. In this paper, we refer to different mechanismsproposed to explain the pathogenesis by previous authors and discussthese theories in relation to our own experimental results. We used ananimal model with lipoproteinlipase deficiency to study thisprogression. In these animals, severe hypertriglyceridemia can beinduced by feeding a high-fat diet, which in turn induces thedevelopment of severe pancreatitis. By sacrificing animals at differenttimes, it was possible with the use of light- and electron microscopy tomonitor the development of acute pancreatitis from the earliestdetectable changes to the most advanced, full-blown stages.

In man, acute pancreatitis in an initial mild stage is not anindication for pancreatic surgery, meaning that biopsies cannot easilybe obtained [1]. In patients with severe acute pancreatitis, the pancreas

is typically grossly pathological and in fatal cases the gland is changedby rapid autolysis and postmortem changes. Animal models such asours therefore provide the only useful way of studying thedevelopment of the disease. At the moment, our mink model withlipoprotein lipase deficiency appears to be the only known animalmodel in which hypertriglyceridemia causes pancreatitis.

Hypertriglyceridemia has been estimated to cause around 7% to10% of the total number of cases of acute pancreatitis in man [2]. Gallstones and alcoholism are other well-known causes. Pancreatitis is alsoknown to be associated with conditions involving poorly regulated fatmetabolism, such as diabetes and obesity [2]. A delectable dinner witha combination of alcohol and delicious, fatty food can provoke a severehypertriglyceridemia in predisposed persons.

The diagnosis can easily be missed. A hypertriglyceridemia presentin the initial stage of pancreatitis, can rapidly subside in patients withacute abdominal pain. They lose appetite, and after admission to ahospital a fasting state is prescribed. Blood triglyceride concentrationshould therefore be measured as early as possible if one is to revealhypertriglyceridemia as the cause of the disease.

Previous theories proposed to explain the pathogenesisSeveral theories have been proposed to explain the pathogenesis of

pancreatitis in hypertriglyceridemia.

Christophersen B, et al., Pancreat Disord Ther 2014, 4:3DOI: 10.4172/2165-7092.1000147

Pancreat Disord TherISSN:2165-7092 PDT, an open access

Volume 4 • Issue 3 • 1000147

Pancreatic Disorders & Therapy Panc

reati

c Disorders & Therapy

ISSN: 2165-7092

Review Open Access

1. A commonly accepted hypothesis was suggested by Havel as earlyas 1969 [3]. In short, excessive amounts of circulating triglyceride-richlipoprotein could be hydrolyzed by inappropriately-activatedpancreatic lipase. Free fatty acids (FFA) could then be liberated intoxic concentrations, causing cell damage.

2. Another theory favors plasma hyperviscosity due to elevatedconcentrations of chylomicrons as the underlying pathophysiologicalprinciple. The hyperviscosity is reputed to lead to ischemia andacidosis in pancreatic capillaries [4,5].

3. Other theories focus on a role of oxidative stress in thepathogenesis of hypertriglyceridemia-induced pancreatitis. Thesuggestion is that free radicals, which are a normal product of cellularmetabolism, are produced in quantities exceeding the capacity ofradical-scavenging mechanisms to absorb them. Contributing factorscould include a relative deficiency of vitamin E, selenium or otherunknown antioxidants [6-9].

Recent results using an unusual animal model: We used mink withlipoprotein lipase deficiency to study the mechanism of thepancreatitis. Blood samples were routinely drawn from juvenilefarmed mink to diagnose a serious, infectious plasmacytosis. Duringthe course of this investigation, it was accidently discovered that someindividuals had severe hypertriglyceridemia [10,11]. Feed of high fat

content is often used on mink farms to obtain high fur quality atreasonable feed cost. It was found that affected individuals werehomozygotic for lipoprotein lipase deficiency and the defect in thelipoprotein lipase gene was characterized [12]. This seems to be theonly known animal model in which pancreatitis can be induced byhypertriglyceridemia. Another animal model in cats with lipoproteinlipase deficiency has been extensively studied, but these cats do notdevelop pancreatitis [13].

By systematically breeding heterozygotes, we were able to obtain asufficient number of homozygotes for further studies. As soon as thepups were weaned, blood samples could be drawn, homozygotes couldbe identified and their fat metabolism could be studied. By sacrificinganimals at different time points relative to the development ofpancreatitis, the gross anatomy, light microscopy and electronmicroscopy of both normal and progressively more damagedpancreases could be studied. All homozygotic animals testeddeveloped pancreatitis at an early age. Later, fatty liver andsplenomegaly were common findings.

The earliest detectable changes in the pancreas occurred in themitochondria of exocrine cells. Light microscopy showed multiplecytoplasmatic vacuoles in exocrine cells in pancreatic acini (Figure 1b)as compared to normal cells (Figure 1a).

Figure 1: Semithin, toluidine blue stained section of pancreas from control mink kit (1a) and lipoprotein-lipase (LPL) deficient mink kit withgrossly normal pancreas (1b). 1a; Normal exocrine cells with abundant zymogen granules. 1b; Vacuolization and reduced amount of zymogengranules in exocrine cells in pancreas from a LPL-deficient mink kit.

This was followed by multicellular necrosis and loss of exocrinecells (Figure 2).

Citation: Christophersen B, Sorby R, Nordstoga K (2014) Studies of the Mechanisms Causing Pancreatitis in Severe Hypertriglyceridemia.Pancreat Disord Ther 4: 147. doi:10.4172/2165-7092.1000147

Page 2 of 5

Pancreat Disord TherISSN:2165-7092 PDT, an open access

Volume 4 • Issue 3 • 1000147

Figure 2: Light microscopy section from a lipoprotein lipasedeficient mink kit with grossly normal pancreas. There is a focus ofmulticellular necrosis of the enzyme producing exocrine cells, withpersistence of intercalated ducts and no inflammatory response.H&E stained section.

Electron microscopy showed that the cytoplasmatic vacuolation wascaused by swelling of mitochondria with loss of crista (Figure 3).

Figure 3: Electron micrograph of exocrine cells from a lipoproteinlipase deficient mink kit. Exocrine cells are packed with well-preserved rough endoplasmic reticulum. There is ballooning ofmitochondria, and an obvious damage of membranes. In the lowerpart, a myelin body is seen.

In some cases the destroyed mitochondrial membranes formedlamellar myelin-like bodies (Figure 4).

Figure 4: Electron micrograph of an exocrine cell from alipoprotein lipase deficient mink kit at a higher magnification,showing a lamellar mitochondrial myelin body. Remnants of cristaesurrounded by flocculent material are seen in the upper right partof the mitochondrion.

In contrast, the other cellular structures were well preserved at thisearly stage of cellular destruction, most notably the roughendoplasmatic reticulum (Figures 3 and 4). Also indicating that thecellular destruction starts inside the cells is the finding that intactdesmosomes were present (Figure 5).

Figure 5: Electron micrograph of exocrine cells from a lipoproteinlipase deficient mink kit showing an acinus with dilatation oflumen and somewhat stunted and distorted microvilli. Balloonedmitochondria, containing membrane remnants at upper and lowerleft. Numerous zymogen granules are present, and desmosomeslook intact (arrows).

Desmosomes are structures that anchor the outer cell membranesof neighboring cells to each other. Results of light- and electronmicroscopy with the focus on veterinary pathology have beenpublished by us in the Journal of Comparative Pathology [14]. As wellknown, the rough endoplasmic reticulum, with its abundantribosomes, is a prominent structure of the exocrine cells because of thesynthesis of digestive protein enzymes such as lipase, trypsin, amylase

Citation: Christophersen B, Sorby R, Nordstoga K (2014) Studies of the Mechanisms Causing Pancreatitis in Severe Hypertriglyceridemia.Pancreat Disord Ther 4: 147. doi:10.4172/2165-7092.1000147

Page 3 of 5

Pancreat Disord TherISSN:2165-7092 PDT, an open access

Volume 4 • Issue 3 • 1000147

and many others. The protein synthesis in the exocrine pancreas isvery active due to its production of digestive proenzymes.

Why should the cellular damage start in the mitochondria?Mitochondria are known to be the major source of cellular free

radicals liberated by the electron transport chain. An excess of FFA inthe cells, exceeding the capacity of intracellular fatty acid binding-proteins, can derange mitochondrial function. FFA can uncouple therespiratory electron transport chain and thus liberate free radicals [15].The hydrophobic end of FFA can penetrate into the lipid doublemembranes and disturb the membrane structure required for therespiratory electron transport chain [16].

How does our finding of an initial, selective degeneration of themitochondria compare with previous theories of the pathogenesis ofhypertriglyceridemic pancreatitis? According to Havel, pancreaticlipase can be released into the vascular bed of the pancreas, andhydrolyze triglycerides in triglyceride-rich lipoproteins when these arepresent in excessive amounts [3]. The very high concentrations of FFAthus formed will exceed the binding capacity of plasma albumin. FFAwill self-aggregate, forming micellar structures with detergentproperties. These FFA micelles will attack platelets, the vascularendothelium and, finally, acinar cells, producing ischemia andpancreatic injury. The resultant ischemia then creates an acidicenvironment, which further enhances FFA toxicity.

We, however, found no signs of endothelial injury, plateletaggregation or ischemia in the earliest stages, as proposed by Havel[3]. Instead we found an extensive initial mitochondrial degeneration.

Both Havel's explanation and our findings of an initialmitochondrial degeneration are, however, in agreement with the viewthat an excessive concentration of FFA plays a key role on thepathogenesis of pancreatitis induced by hypertriglyceridemia. Twodifferent sources of the excess of FFA can however be discussed.

1. Triglyceriderich lipoproteins in the vascular bed could be thesource after hydrolysis by pancreatic lipase.

2. Lipoproteins internalized by receptor mediated uptake in theexocrine pancreas cells could be the source.

As proposed by Havel, it is possible that these FFAs could beliberated by pancreatic lipase acting on triglyceride-rich lipoproteinsin the vascular bed of the gland. This would require two conditions tobe fulfilled. It would require that the lipase proenzyme produced bythe exocrine pancreas cells can be activated in situ in the gland itself orin its vascular bed. This activation of the enzyme is traditionallyconsidered to take place in the duodenum. It would also require thatVLDL and/or chylomicron can serve as substrates for pancreas lipase.Although this seems to be likely, it has not been directly shownexperimentally.

If the selective mitochondrial degeneration is caused by FFAliberated in the pancreatic vascular bed, it would imply that 1) FFA areformed in excess of the binding capacity of albumin and 2) afteruptake in the acinar cells the FFA concentration should also exceed thebinding capacity of intracellular fatty acid binding proteins [14]. Thisconcentration of FFA could then be toxic to the mitochondria while atthe same time not causing visible damage to the external cellmembrane or other intracellular membranes such as the endoplasmicreticulum. This may be the case if the mitochondria with their electrontransport chain are more sensitive to an excess of FFA than are otherlipid membranes such as the vascular endothelium, platelets and the

external cell membrane of acinar cells and other intracellularmembranes.

As an alternative to Havel's hypothesis, it can be hypothesized thatan excess of FFA can be liberated by intracellular hydrolysis oftriglyceride-rich lipoproteins after uptake in the acinar pancreas cells[11]. It is well known that chylomicron and VLDL can be taken up asintact particles by cells in most tissues when the concentration oftriglyceride-rich lipoproteins is very high. We found that also inlipoprotein lipase-deficient animals, the hypertriglyceridemia subsideson fasting or on a low-fat diet [10]. As is well known, hepatocytes havean abundance of such lipoprotein receptors. Since the pancreas andthe liver both originate from the same structures duringembryogenesis, it may also be postulated that the pancreas is rich inreceptors for triglyceride-rich lipoproteins.

Hyperviscosity: As stated it has been proposed thathypertriglyceridemia could cause hyperviscosity in the pancreaticvascular bed and thus cause cellular ischemia [4,5]. Our finding of aninitial mitochondrial degeneration does not support the theory thathyperviscosity plays an important role in the pathogenesis of this typeof pancreatitis.

Oxidative stress: As mentioned above, oxidative stress has beenproposed to play a role in inducing pancreatitis inhypertriglyceridemia [6]. The causes of the imbalance between freeradicals and scavenging mechanisms in hypertriglyceridemia have notbeen established. Our finding of an initial mitochondrial degenerationagrees with the view that oxidative stress is important in thepathogenesis. In our view it is an excess of FFA that leads to oxidativestress in the mitochondria.

Prevention and treatmentTo prevent or treat hypertriglyceridemia is the primary goal in

patients suffering from this type of pancreatitis. Inhypertriglyceridemia caused by high levels of VLDL and ofchylomicron (Fredrichson's type V), dietary change, eicosapentaenoicacid supplementation and specific drug therapy can all be beneficial.In the rare, congenital errors with hyperchylomicronemia(Fredrichson's type I), treatment strategies are more demanding, andmay include plasmapheresis.

Antioxidants such as vitamin E have been tried in a few patients inthe acute stage of pancreatitis, although the low number of patientsincluded has not provided conclusive results [17]. In one study threepatients with frequent episodes of pancreatitis due tohyperchylomicronemia caused by lipoprotein lipase deficiency weretreated with high doses of antioxidants (selenium, carotene andvitamin E) [6]. After this regime was started, the number and severityof episodes of pancreatitis were markedly reduced.

Our findings of an initial mitochondrial degeneration, mostprobably caused by free radicals leading to oxidative stress, support theview that antioxidants may have a role in the prevention of recurrenthypertriglyceridemia-induced pancreatitis. In the future, clinical trialswith and without high doses of antioxidants should be carried out inpatients suffering from recurrent hypertriglyceridemic pancreatitis.

References1. Uhl W, Warshaw A, Imre C, Bassi C, McCay CJ, et al. (2002) IAP

guidelines of the surgical management of acute pancreatitis.Pancreatology 2: 565-573.

Citation: Christophersen B, Sorby R, Nordstoga K (2014) Studies of the Mechanisms Causing Pancreatitis in Severe Hypertriglyceridemia.Pancreat Disord Ther 4: 147. doi:10.4172/2165-7092.1000147

Page 4 of 5

Pancreat Disord TherISSN:2165-7092 PDT, an open access

Volume 4 • Issue 3 • 1000147

2. Ewald N, Hardt PD, Kloer H-U (2009) Severe hypertriglyceridemia andpancreatitis: presentation and management. Current Opinion inLipidology 20: 497-504.

3. Havel RJ (1969) Pathogenesis, differentiation and management ofhypertriglyceridemia. Adv Inter Med 15: 117-145.

4. Saharia P, Margolis S, Zuidema MD, Cameron JL (1977) Acutepancreatitis with hypertriglyceridemia: studies with an isolated perfusedcanine pancreas. Surgery 82: 60-67.

5. Kimura W, Mossner J (1969) Role of hypertriglyceridemia in thepathogenesis of experimental acute pancreatitis in rats. Int J Pancreatol20: 177-184.

6. Heaney AP, Sharer N, Rameh B, Braganza JM, Durrington PN (1999)Prevention of Recurrent Pancreatitis in Familial Lipoprotein LipaseDeficiency with High-Dose Antioxidant Therapy. J of ClinicalEndocrinology & Metabolism 84: 1203-1205.

7. Zhang X, Cui Y, Fang L, Li F (2008) Chronic high-fat diets induce oxideinjuries and fibrogenesis of pancreatic cells in rats. Pancreas 37: 31-38.

8. McCoy MA, McLoughlin MF, Rice DA, Kennedy DG (1994) Pancreasdisease in Atlantic salmon (Salmo salar) and vitamin E supplementation.Comp Biochem Physiol 109: 905-912.

9. Whitacre ME, Combs GF, Combs SB, Parker RS (1987) Influence ofdiatary vitamin E on nutritional pancreatic atrophy in selenium-deficientchicks. J Nutr 117: 460-467.

10. Christophersen B, Nordstoga K, Shen Y, Olivecrona T, Olivecrona G(1997) Lipoprotein lipase deficiency with pancreatitis in mink:

biochemical characterization and pathology. J of Lipid Research 38:837-846.

11. Savonen R, Nordstoga K, Christophersen B, Lindberg A, Shen Y, et al.(1999) Chylomicron metabolism in an animal model forhyperlipoproteinemia type I. J of Lipid Research 40:1336-1346.

12. Lindberg A, Nordstoga K, Christophersen B, Savonen R, Van T, et al.(1998) A mutation in the liprotein lipase gene associated withhyperlipoproteinemia type I in mink. International Journal of MolecularMedicine 1: 529-538.

13. Ginzinger DG, Lewis ME, Ma YH, Jones BR, Liu G, et al. (1996) Amutation in the lipoprotein lipase gene is the molecular basis ofchylomicronemia in a colony of domestic cats. J Clin Invest 97:1257-1266.

14. Nordstoga K, Christophersen B, Ytrehus B, Espenes A, Osmundsen H, etal. (2006) Pancreatitis associated with hyperlipoproteinemia Type I inmink (Mustela vison): Earliest detectable changes occur in mitochondriaof exocrine cells. J Comp Pathol 134: 320-328.

15. Rial E, Rodriguez-Sanches L, Gallardo-Vara E, Zaragoza P, Moyano E, etal. (2010) Lipotoxicity, fatty acid uncoupling ans mitochondrial carrierfunction. Biochim Biophys Acta 1797: 800-806.

16. Scow RO, Blanchette-Mackie EJ (1991) Transport of fatty acids andmonoacylglycerols in white and brown adipose tissue. Brain researchBulletin 27: 487-491.

17. Miller JP (2000) Serum triglycerides, the liver and the pancreas. CurrentOpinion in Lipidology 11: 377-382.

Citation: Christophersen B, Sorby R, Nordstoga K (2014) Studies of the Mechanisms Causing Pancreatitis in Severe Hypertriglyceridemia.Pancreat Disord Ther 4: 147. doi:10.4172/2165-7092.1000147

Page 5 of 5

Pancreat Disord TherISSN:2165-7092 PDT, an open access

Volume 4 • Issue 3 • 1000147